scholarly journals Mannose-specific endocytosis receptor of alveolar macrophages: demonstration of two functionally distinct intracellular pools of receptor and their roles in receptor recycling.

1982 ◽  
Vol 92 (2) ◽  
pp. 417-424 ◽  
Author(s):  
C Tietze ◽  
P Schlesinger ◽  
P Stahl
Keyword(s):  
Cell Surface ◽  
Ammonium Chloride ◽  
Alveolar Macrophages ◽  
Binding Activity ◽  
Dissociation Rate ◽  
Receptor Recycling ◽  
Surface Binding ◽  
Receptor Ligand ◽  
Receptor Mediated Endocytosis ◽  
Weak Bases

Receptor-mediated endocytosis of rat preputial beta-glucuronidase and the glycoconjugate mannose-BSA by rat alveolar macrophages is inhibited by chloroquine and ammonium chloride. We have previously reported that these drugs cause a loss of cell surface binding activity and that they do not inhibit internalization of receptor ligand complexes when incubated with cells at 37 degrees C. In this report we more clearly delineate the intracellular site of weak base inhibition of receptor recycling and the mechanism of that inhibition. From our analysis of the kinetics of ligand transport we conclude that there are two functionally distinct intracellular pools of receptor. One of these, the cycling pool, is not sensitive to the presence of weak bases, and receptor-ligand complexes return from this pool to the cell surface intact. The second pool is responsible for the time-dependent intracellular delivery of ligand to acid vesicles, which is inhibited by weak bases. Chloroquine and ammonium chloride appear to inhibit the dissociation of receptor-ligand complexed in this second pool and thereby the production of free receptors for the continuation of receptor-mediated endocytosis. We examine the internalization and binding of ligand in normal and paraformaldehyde-treated cells and find that these are strongly affected by pH. In particular, the dissociation rate of receptor ligand complexes is enhanced greater than 7.5 fold by lowering the medium pH from 7 to 6. From these results we propose that weak bases raise the pH of acid intracellular compartments, slowing the rate of receptor-ligand dissociation and thereby reducing the cellular pool of free receptors available for further uptake of ligand. In addition, we demonstrate that receptor-ligand complexes cannot return to the cell surface from the amine-sensitive (acid) intracellular pool that led us to call this the nonreleasable pool. This final observation indicates that receptor movements through these two pools are functionally distinct processes.

scholarly journals Monensin inhibits recycling of macrophage mannose-glycoprotein receptors and ligand delivery to lysosomes

1984 ◽  
Vol 220 (3) ◽  
pp. 665-675 ◽  
Author(s):  
T Wileman ◽  
R L Boshans ◽  
P Schlesinger ◽  
P Stahl
Keyword(s):  
Cell Surface ◽  
Specific Binding ◽  
Binding Activity ◽  
Surface Binding ◽  
Binding Studies ◽  
Receptor Ligand ◽  
Lysosomal Fraction ◽  
Cell Surface Binding ◽  
Monensin A ◽  
Proton Movement

Binding studies with cells that had been permeabilized with saponin indicate that alveolar macrophages have an intracellular pool of mannose-specific binding sites which is about 4-fold greater than the cell surface pool. Monensin, a carboxylic ionophore which mediates proton movement across membranes, has no effect on binding of ligand to macrophages but blocks receptor-mediated uptake of 125I-labelled beta-glucuronidase. Inhibition of uptake was concentration- and time-dependent. Internalization of receptor-bound ligand, after warming to 37 degrees C, was unaffected by monensin. Moreover, internalization of ligand in the presence of monensin resulted in an intracellular accumulation of receptor-ligand complexes. The monensin effect was not dependent on the presence of ligand, since incubation of macrophages with monensin at 37 degrees C without ligand resulted in a substantial decrease in cell-surface binding activity. However, total binding activity, measured in the presence of saponin, was much less affected by monensin treatment. Removal of monensin followed by a brief incubation at pH 6.0 and 37 degrees C, restored both cell-surface binding and uptake activity. Fractionation experiments indicate that ligands enter a low-density (endosomal) fraction within the first few minutes of uptake, and within 20 min transfer to the lysosomal fraction has occurred. Monensin blocks the transfer from endosomal to lysosomal fraction. Lysosomal pH, as measured by the fluorescein-dextran method, was increased by monensin in the same concentration range that blocked ligand uptake. The results indicate that monensin blockade of receptor-mediated endocytosis of mannose-terminated ligands by macrophages is due to entrapment of receptor-ligand complexes and probably receptors in the pre-lysosomal compartment. The inhibition is linked with an increase in the pH of acid intracellular vesicles.

The ovine pancreatic protein which binds insulin-like growth factor binding protein-3 is procarboxypeptidase A

1996 ◽  
Vol 150 (1) ◽  
pp. 51-56 ◽  
Author(s):  
P J Fowke ◽  
S C Hodgkinson
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Binding Protein ◽  
Binding Activity ◽  
Insulin Like Growth Factor ◽  
Surface Binding ◽  
Factor Binding ◽  
Amino Terminal ◽  
Igf I ◽  
Igfbp 3

Abstract Insulin-like growth factor binding protein-3 (IGFBP-3) is known to modulate the actions of insulin-like growth factors (IGF)-I and -II at the level of the cell. Proposed mechanisms include association of IGFBP-3 with cell surface proteoglycan, with cell surface binding proteins, proteolysis and/or internalization of IGFBP-3. In previous studies we have characterized a protein of 40 kDa in extracts of ovine pancreas and muscle which binds IGFBP-3 on ligand blot analyses. This paper reports the identity of the pancreatic species as procarboxypeptidase A (peptidyl-l-amino acid hydrolase, E.C. 3.4.17.1; proCPA). Identity was established by amino terminal sequence analysis, binding studies with pure bovine carboxypeptidase A (CPA) and observations that the binding activity was present in pancreatic secretions consistent with the role of proCPA as a secretory zymogen. The binding activity was inhibited by unlabelled IGFBP-3 at high doses (10 μg/ml) and reduced but not abolished by preincubation of 125I-IGFBP-3 with excess IGF-I. Digestion of 125I-IGFBP-3 with mature CPA produced a 26 kDa product. Modification of IGFBP-3 by CPA or binding to proCPA may provide a mechanism for modulation of IGFBP activity and hence IGF action. Journal of Endocrinology (1996) 150, 51–56

Abstract 5: ApoAI Binds to Phosphatidylinositol 4,5 Bisphosphate (PIP2), Which is Exposed on the Cell Surface by Novel PIP2 Floppase Activity of ABCA1, and Promotes Cholesterol Efflux

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Kailash Gulshan ◽  
Gregory Brubaker ◽  
Stanley Hazen ◽  
Jonathan Smith
Keyword(s):  
Cell Surface ◽  
Cholesterol Efflux ◽  
Binding Activity ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Ph Domain ◽  
Surface Binding ◽  
Raw264.7 Macrophages ◽  
Lipid Species ◽  
Phosphatidylinositol Phosphates

Introduction: ApoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse cholesterol transport, but the mechanism of HDL assembly is unknown. Objective: To determine role of PIP2 in ApoAI-ABCA1 mediated nascent HDL assembly. Methods and Results: To determine which lipid species are directly bound by apoAI, a lipid-protein overlay assay was performed using PIPstrips and sphingostrips from Echelon Biosciences. ApoAI bound directly to phosphatidylinositol phosphates (PIPs) rather than PC, PS, PE, SM or cholesterol. This interaction was not solely electrostatic, as PIP3, the most negative charged PIP had less apoAI binding than various PIP2 species. ApoAI did not bind to other charged lipids such as sphingosine 1-P, PI(3)-P, PI(4)-P, and PI(5)-P. Surface plasmon resonance assays using immobilized apoAI or PIP2 were performed, showing that apoAI interacted reversibly with PI(4,5)P2 in a dose dependent manner at the nM concentration range. The central domain of apoAI (residues 44-185) was sufficient for PIP2 binding. In RAW264.7 macrophages, depletion of cellular PIPs by treatments with PI3K inhibitor or PTEN inhibitor decreased cholesterol efflux by 45±1.4% and 41±6.4% respectively (p<0.005). Degradation of cell surface PIP2 via treatment with PI specific PLC decreased cell surface binding of apoAI by 37±6% and cholesterol efflux by 48±8% (p<0.005). In mouse bone-marrow derived macrophages, cholesterol efflux to apoAI was increased ~47% by GRIP (PIP2 binding PH domain of PLCδ) and decreased 41±8% by PTEN inhibition (p<0.005). Liposomes made with POPC:POPS:FC:PIP2 (65:20:10:5 mole ratio) were solubilized by apoAI at 37 o C, pH5, but apoAI had little activity using similar liposomes without PIP2 . ABCA1 expression led to 2-fold higher cell surface PIP2, assayed by flow cytometry using a PIP2 specific antibody. RAW cells stably transfected with PIP2 binding domain 2X PH-PLCδ-eGFP showed enrichment of this PIP2 reporter on the plasma membrane, while expression of ABCA1 led to partial redistribution to cytoplasm, consistent with ABCA1 having PIP2 floppase activity. Conclusions: ApoAI has novel PIP2 binding activity, ABCA1 is a novel PIP2 floppase, and cell surface PIP2 is required for optimal cholesterol acceptor activity of apoAI.

scholarly journals Evidence that the thyrotropin-releasing hormone receptor and its ligand are recycled dissociated from each other

1995 ◽  
Vol 306 (1) ◽  
pp. 107-113 ◽  
Author(s):  
C P Petrou ◽  
A H Tashjian
Keyword(s):  
Cell Surface ◽  
Hormone Receptor ◽  
Thyrotropin Releasing Hormone ◽  
Cell Surface Receptor ◽  
Acidic Ph ◽  
Receptor Recycling ◽  
Bafilomycin A1 ◽  
Receptor Ligand ◽  
Surface Receptor ◽  
Receptor Pool

We have examined the trafficking of the thyrotropin-releasing hormone receptor (TRHR) and its ligand, after TRHR-TRH internalization in rat pituitary GH4C1 cells. After rapid ligand-induced receptor sequestration, the cell surface receptor pool was replenished. Replenishment was insensitive to inhibition of protein synthesis and was dependent on the duration of internalization; therefore, the replenished receptors were not newly synthesized but recycled. The total amount of recycled receptors decreased with increasing internalization time, resulting in only partial replenishment of the cell-surface receptor pool after prolonged incubation with ligand. Thus, in addition to a receptor recycling pathway, a non-cycling route exists for TRHR sorting; this route became dominant with increasing internalization periods. TRHR entry into these pathways was not determined by the affinity of the receptor-ligand interaction, because the extent of receptor recycling was similar after TRH- and methyl-TRH (MeTRH)-induced internalization. Unlike results with the TRHR, the TRH recycling pool was not depleted by the noncycling pathway. After multiple rounds of [3H]MeTRH internalization, the amount of cell-associated radioactivity increased with increasing internalization time due to accumulation of the ligand or its metabolites in a non-cycling pathway, but the absolute amount of recycled ligand remained constant after short or long internalization times. The difference in the proportion of TRHR and MeTRH that were diverted into a noncycling pathway indicated intracellular dissociation of the internalized TRHR-TRH complex. Dissociation of the internalized TRHR-TRH complex was dependent on the acidic pH in an intracellular compartment. Although extracellular acidic pH did not enhance cell-surface receptor-ligand (RL) dissociation, bafilomycin A1 inhibited both receptor and ligand recycling. We conclude that the TRHR-TRH system is unique among recycling receptors because, after RL sequestration, the TRHR-TRH complex becomes dissociated intracellularly via a bafilomycin A1-sensitive, acidic pH-dependent mechanism, and both the unoccupied TRHR and TRH recycle disassociated from each other.

scholarly journals Is movement of mannose 6-phosphate-specific receptor triggered by binding of lysosomal enzymes?

1987 ◽  
Vol 104 (6) ◽  
pp. 1735-1742 ◽  
Author(s):  
T Braulke ◽  
C Gartung ◽  
A Hasilik ◽  
K von Figura
Keyword(s):  
Cell Surface ◽  
Lysosomal Enzymes ◽  
Free Cell ◽  
Cell Surface Receptors ◽  
Receptor Ligands ◽  
Receptor Ligand ◽  
Surface Receptors ◽  
Weak Bases ◽  
Specific Receptors ◽  
Mannose 6 Phosphate

Mannose 6-phosphate-specific receptors with an apparent molecular mass of 215,000 are present in fibroblasts at the cell surface and in intracellular membranes. The cell surface receptors mediate endocytosis of exogenous lysosomal enzymes and exchange with the intracellular receptors, which function in the sorting of endogenous lysosomal enzymes. In the present study, several methods independent of receptor ligands were designed in order to examine the exchange of receptors under conditions where receptor-ligand complexes do not dissociate (weak bases and monensin) or where receptor-ligand complexes are not formed due to absence of endogenous ligands as a result of inhibition of protein synthesis. Weak bases and monensin reduce the concentration of receptors at the cell surface by 20-30% and free cell surface receptors were replaced by occupied receptors. The latter continued to be exchanged with internal ligand-occupied receptors and the rates of the exchange were similar to the control values. The exchange of receptors between the cell surface and internal membranes was also not affected when the receptor ligands were depleted from the transport compartments by treating the cells with cycloheximide for up to 10 h. We conclude from these results that movement of mannose 6-phosphate-specific receptors along the endocytosis and sorting pathways is constitutive and not triggered by binding or dissociation of ligands.

scholarly journals Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

1982 ◽  
Vol 92 (3) ◽  
pp. 634-647 ◽  
Author(s):  
PL Zeitlin ◽  
AL Hubbard
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Binding Sites ◽  
Rat Hepatocytes ◽  
Surface Binding ◽  
Isolated Rat Hepatocytes ◽  
Electron Microscopic Autoradiography ◽  
Surface Receptors ◽  
Receptor Mediated Endocytosis ◽  
Isolated Rat

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.

scholarly journals Identification of a novel cell attachment domain in the HIV-1 Tat protein and its 90-kDa cell surface binding protein.

1993 ◽  
Vol 268 (7) ◽  
pp. 5279-5284
Author(s):  
B.S. Weeks ◽  
K. Desai ◽  
P.M. Loewenstein ◽  
M.E. Klotman ◽  
P.E. Klotman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Attachment ◽  
Binding Protein ◽  
Tat Protein ◽  
Surface Binding ◽  
Cell Surface Binding ◽  
Hiv 1

scholarly journals Cell surface-binding sites for progesterone mediate calcium uptake in human sperm.

1991 ◽  
Vol 266 (28) ◽  
pp. 18655-18659 ◽  
Author(s):  
P.F. Blackmore ◽  
J. Neulen ◽  
F. Lattanzio ◽  
S.J. Beebe
Keyword(s):  
Cell Surface ◽  
Binding Sites ◽  
Calcium Uptake ◽  
Human Sperm ◽  
Surface Binding ◽  
Cell Surface Binding

Perfluorocarbon attenuates response of concanavalin A-stimulated mononuclear blood cells without altering ligand-receptor interaction

2004 ◽  
Vol 287 (1) ◽  
pp. L210-L216 ◽  
Author(s):  
Dirk Haufe ◽  
Thomas Luther ◽  
Matthias Kotzsch ◽  
Lilla Knels ◽  
Thea Koch
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Blood Cells ◽  
Cell Activation ◽  
Receptor Interaction ◽  
Surface Binding ◽  
Cellular Activation ◽  
Flow Cytofluorometry ◽  
Anti Inflammatory ◽  
Mononuclear Blood Cells

Intrapulmonary application of perfluorocarbons (PFC) in acute lung injury is associated with anti-inflammatory effects. A direct impact on leukocytic function may be involved. To further elucidate PFC effects on cellular activation, we compared in an in vitro model the response of concanavalin A (ConA)-stimulated lymphocytes and monocytes exposed to perfluorohexane. We hypothesized that perfluorohexane attenuates the action of the lectin ConA by altering stimulant-receptor interaction on the cell surface. Mononuclear blood cells were stimulated by incubation with ConA in the presence of different amounts of perfluorohexane. The response of lymphocytes and monocytes was determined by means of IL-2 secretion and tissue factor (TF) expression, respectively. The influence of perfluorohexane on cell-surface binding of fluorescence-labeled ConA was studied using flow cytofluorometry and fluorescence microscopy. Perfluorohexane itself did not induce a cellular activation but significantly inhibited both monocytic TF expression and, to a far greater extent, IL-2 secretion of ConA-stimulated mononuclear blood cells. The effect of perfluorohexane was due neither to an alteration of cell viability nor to a binding of the stimulant. The amount of cell surface-bound ConA was not altered by perfluorohexane, and the overall pattern of ConA receptor rearrangement did not differ between controls and treated cells. In the present study, we provide further evidence for an anti-inflammatory effect of PFC that might be beneficial in states of pulmonary hyperinflammation. A PFC-induced alteration of stimulant-receptor interaction on the surface membrane does not seem to be the cause of attenuated cell activation.

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